US8241622B2ExpiredUtilityA1

Adeno-associated virus vectors with intravector heterologous terminal palindromic sequences

84
Assignee: ENGLEHARDT JOHN FPriority: Jul 13, 2001Filed: Apr 30, 2004Granted: Aug 14, 2012
Est. expiryJul 13, 2021(expired)· nominal 20-yr term from priority
Inventors:John EnglehardtZiying Yan
A61P 31/12C12N 15/86A61K 48/00A61K 48/0008C12N 2750/14143C12N 2750/14145C12N 2810/60A61P 35/00A61P 37/06A61P 31/04
84
PatentIndex Score
52
Cited by
272
References
58
Claims

Abstract

The invention provides recombinant AAV vectors, at least one of which has heterologous terminal palindromic sequences, and methods of using those vectors.

Claims

exact text as granted — not AI-modified
1. A vector set comprising at least two recombinant adeno-associated virus (rAAV) vectors, wherein a first rAAV vector has two inverted terminal repeats (ITRs) that are heterologous, and a second rAAV vector has two ITRs, at least one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the first rAAV vector and the second rAAV vector to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the second rAAV vector is formed at an increased efficiency relative to a control rAAV vector set comprising two rAAV vectors with four homologous ITRs, the two ITRs of the second rAAV vector may be homologous or heterologous, and wherein the heterologous ITRs are from different AAV serotypes. 
     
     
       2. The set of  claim 1  wherein the first rAAV vector or the second rAAV vector has a portion of an open reading frame for a gene product, optionally linked to at least one heterologous transcriptional regulatory element, and a splice donor site 3′ to the portion of the open reading frame, wherein the second rAAV vector or the first rAAV vector comprises the remainder of the open reading frame with a splice acceptor site 5′ to the remainder of the open reading frame. 
     
     
       3. The set of  claim 2  wherein the at least one heterologous transcriptional regulatory element is a promoter. 
     
     
       4. The set of  claim 2  wherein the at least one heterologous transcriptional regulatory element comprises a promoter and an enhancer. 
     
     
       5. The set of  claim 1  wherein the first rAAV vector or the second rAAV vector comprises an enhancer. 
     
     
       6. The set of  claim 5  wherein the second rAAV vector or the first rAAV vector comprises an open reading frame for a gene product. 
     
     
       7. The set of  claim 6  wherein the second rAAV vector or the first rAAV vector further comprises a promoter operably linked to the open reading frame for the gene product. 
     
     
       8. The set of  claim 2  or  6  wherein the gene product is a therapeutic gene product. 
     
     
       9. The set of  claim 2  or  6  wherein the gene product is a catalytic RNA. 
     
     
       10. The set of  claim 2  or  6  wherein the gene product is a prophylactic gene product. 
     
     
       11. The set of  claim 2  or  6  wherein the gene product is a polypeptide or peptide. 
     
     
       12. The set of  claim 1  wherein the first rAAV vector or the second rAAV vector comprises a first promoter. 
     
     
       13. The set of  claim 12  wherein the second rAAV vector or the first rAAV vector comprises an open reading frame for a gene product. 
     
     
       14. The set of  claim 13  wherein the second rAAV vector or the first rAAV vector further comprises a second promoter operably linked to the open reading frame for the gene product. 
     
     
       15. The set of  claim 1 , wherein the first rAAV vector comprises an open reading frame for a first gene product and the second rAAV vector comprises an open reading frame for a second gene product, wherein the first and the second gene products are different and the open reading frames are each individually operably linked to an expression control sequence. 
     
     
       16. The set of  claim 1  wherein the 3′ ITR in the first rAAV vector or the second rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 or AAV-8 ITR. 
     
     
       17. The set of  claim 1  wherein the 5′ ITR in the first rAAV vector is homologous to the 3′ ITR in the second rAAV vector. 
     
     
       18. The set of  claim 1  wherein the 5′ ITR in the first rAAV vector is heterologous to the 3′ ITR in the second rAAV vector. 
     
     
       19. The set of  claim 1  wherein the 5′ ITR in the first rAAV vector or the second rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 or AAV-8 ITR. 
     
     
       20. The vector set of  claim 1  wherein the ITRs of the second rAAV vector are homologous. 
     
     
       21. The vector set of  claim 1  wherein the ITRs of the second rAAV vector are heterologous. 
     
     
       22. A composition comprising at least two rAAV, the composition comprising:
 a first rAAV having two ITRs that are heterologous, and a second rAAV having two ITRs, at least one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV, wherein after introduction of the first rAAV and the second rAAV to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV and the second rAAV is formed at an increased efficiency relative to two control AAVs comprising four homologous ITRs, wherein the two ITRs of the second rAAV may be homologous or heterologous, and wherein the heterologous ITRs are from different serotypes. 
 
     
     
       23. The composition of  claim 22  further comprising a delivery vehicle. 
     
     
       24. The composition of  claim 23  where the vehicle is a pharmaceutically acceptable carrier. 
     
     
       25. An isolated host cell contacted with a composition comprising a first rAAV vector having two ITRs that are heterologous, and a second rAAV vector having two ITRs, at least one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the composition to the host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the second rAAV vector is formed at an increased efficiency relative to a control rAAV vector set having two rAAV vectors with four homologous ITRs, and wherein the two ITRs of the second rAAV vector may be homologous or heterologous, wherein the heterologous ITRs are from different serotypes. 
     
     
       26. The isolated host cell of  claim 25  which is a mammalian host cell. 
     
     
       27. An isolated host cell contacted with a first rAAV vector having two ITRs that are heterologous, and a second rAAV vector having two ITRs at least one of is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the first rAAV vector and the second rAAV vector to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the second rAAV vector is formed at an increased efficiency relative to a control rAAV vector set having two rAAV vectors with four homologous ITRs, wherein the two ITRs of the second rAAV vector may be homologous or heterologous, and wherein the heterologous ITRs are from different serotypes. 
     
     
       28. The isolated host cell of  claim 27  which is a mammalian host cell. 
     
     
       29. A vector set comprising at least two rAAV vectors, wherein a first rAAV vector has two ITRs that are heterologous, and a second rAAV vector has two ITRs one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the first rAAV vector and the second rAAV vector to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the rAAV vector is formed at an increased efficiency relative to two control rAAV vectors with four homologous ITRs, wherein one ITR in the first rAAV vector or the second rAAV vector is a pseudo ITR having a palindromic DNA sequence with one of SEQ ID Nos. 3, 7, 8, 9, or 10. 
     
     
       30. A vector set comprising at least two rAAVs, wherein the set comprises:
 a) a first rAAV comprising a first recombinant DNA molecule comprising operably linked: 
 i) a first DNA segment comprising a first 5′ ITR; 
 ii) a second DNA segment comprising a non-AAV DNA sequence; and 
 iii) a third DNA segment comprising a first 3′ ITR, wherein the first 5′ ITR is heterologous to the first 3′ ITR; and
 b) a second rAAV comprising a second recombinant DNA molecule comprising operably linked: 
 
 i) a first DNA segment comprising a second 5′ ITR; 
 ii) a second DNA segment comprising a non-AAV DNA sequence which is different than the sequence in the second DNA segment of the first recombinant DNA molecule; and 
 iii) a third DNA segment comprising a second 3′ ITR, wherein the second 5′ ITR is optionally heterologous to the second 3′ ITR, wherein the first 3′ ITR is homologous to the second 5′ ITR, wherein at least one of the second DNA segments comprises an open reading frame encoding a functional gene product or the two second DNA segments together comprise an open reading frame encoding a functional gene product, which homologous ITRs, after infection of a cell with the vector set, are capable of directional intermolecular recombination yielding a chimeric DNA molecule at an increased efficiency relative to control rAAVs having two rAAVs with four homologous ITRs, wherein the chimeric DNA molecule comprises an expression control sequence so that the open reading frame is capable of being transcribed to yield a RNA molecule that encodes the gene product, wherein one ITR in the first rAAV vector or the second rAAV vector is a pseudo ITR having a palindromic DNA sequence with f one of SEQ ID Nos. 3, 7, 8, 9 or 10. 
 
     
     
       31. The vector set of  claim 29  or  30  wherein the pseudo ITR has an imperfect palindomic DNA sequence. 
     
     
       32. The vector set of  claim 29  or  30  wherein the pseudo ITR has SEQ ID NO:3, SEQ ID NO:7or SEQ ID NO:8. 
     
     
       33. A method to transfer DNAs into a host cell, comprising:
 contacting the host cell with a first rAAV vector having two ITRs that are heterologous, and a second rAAV vector having two ITRs one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the first rAAV vector and the second rAAV vector to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the second rAAV vector is formed at an increased efficiency relative to a control rAAV vector set having two rAAV vectors with four homologous ITRs, wherein the heterologous ITRs are from different AAV serotypes. 
 
     
     
       34. The method of  claim 33  wherein the host cell is a mammalian host cell. 
     
     
       35. The method of  claim 33  wherein the first rAAV vector or the second rAAV vector has a portion of an open reading frame for a gene product, optionally linked to at least one heterologous transcriptional regulatory element, and a splice donor site 3′ to the portion of the open reading frame, wherein the second rAAV vector or the first rAAV vector comprises the remainder of the open reading frame with a splice acceptor site 5′ to the remainder of the open reading frame. 
     
     
       36. The method of  claim 35  wherein the at least one heterologous transcriptional regulatory element is a promoter. 
     
     
       37. The method of  claim 35  wherein the at least one heterologous transcriptional regulatory element comprises a promoter and an enhancer. 
     
     
       38. The method of  claim 33  wherein the first rAAV vector or the second rAAV vector comprises an enhancer. 
     
     
       39. The method of  claim 38  wherein the second rAAV vector or the first rAAV vector comprises an open reading frame for a gene product. 
     
     
       40. The method of  claim 39  wherein the second rAAV vector or the first rAAV vector further comprises a promoter operably linked to the open reading frame for the gene product. 
     
     
       41. The method of  claim 35  or  39  wherein the gene product is a therapeutic gene product. 
     
     
       42. The method of  claim 35  or  39  wherein the gene product is a catalytic RNA. 
     
     
       43. The method of  claim 35  or  39  wherein the gene product is a prophylactic gene product. 
     
     
       44. The method of  claim 35  or  39  wherein the gene product is a polypeptide or a peptide. 
     
     
       45. The method of  claim 33  wherein the first rAAV vector or the second rAAV vector comprises a first promoter. 
     
     
       46. The method of  claim 30  or  31  wherein the first 5′ TPS or second 3′ TPS is a native TPS. 
     
     
       47. The method of  claim 46  wherein the second rAAV vector or the first rAAV vector further comprises a second promoter operably linked to the open reading frame for the gene product. 
     
     
       48. The method of  claim 33  wherein the first rAAV vector comprises an open reading frame for a first gene product and the second rAAV vector comprises an open reading frame for a second gene product, wherein the first and second gene products are different. 
     
     
       49. The method of  claim 33  wherein the 3′ ITR in the first rAAV vector or the second rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 or AAV-8 ITR. 
     
     
       50. The method of  claim 33  wherein the 5′ ITR in the first rAAV vector is homologous to the 3′ ITR in the second rAAV vector. 
     
     
       51. The method of  claim 33  wherein the 5′ ITR in the first rAAV vector is heterologous to the 3′ ITR in the second rAAV vector. 
     
     
       52. The method of  claim 33  wherein the 5′ ITR in the first rAAV vector or the second rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 or AAV-8 ITR. 
     
     
       53. A method to transfer and express a functional gene product in a host cell, the method comprising providing a composition comprising a first rAAV having two ITRs that are heterologous, and a second rAAV having two ITRs, at least one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV, wherein after introduction of the first rAAV and the second rAAV to a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV and the second rAAV is formed at an increased efficiency relative to two control rAAVs with four homologous ITRs, the chimeric DNA molecule comprises an expression control sequence operably linked to an open reading frame encoding a functional gene product, wherein the two ITRs of the second rAAV may be homologous or heterologous, wherein the heterologous ITRs are from different serotypes; and contacting the host cell with the composition so that the host cell expresses the functional gene product. 
     
     
       54. The method of  claim 53 , wherein the ITRs of the second rAAV vector are homologous. 
     
     
       55. The method of  claim 53  wherein the ITRs of the second rAAV vector are heterologous. 
     
     
       56. A method to transfer DNAs into a host cell, comprising:
 contacting the host cell with a first rAAV vector has two ITRs that are heterologous, and a second rAAV vector has two ITRs one of which is homologous to and is in the same orientation as one of the ITRs in the first rAAV vector, wherein after introduction of the first rAAV vector and the second rAAV vector to a host or a host cell, a chimeric DNA molecule comprising nucleic acid sequences from the first rAAV vector and the second rAAV vector is formed at an increased efficiency relative to control rAAV vectors having two rAAVs with four homologous ITRs, wherein one ITR in the first rAAV vector or the second rAAV vector is a pseudo ITR having a palindromic DNA sequence with one of SEQ ID Nos. 3,7,8,9 or 10. 
 
     
     
       57. The method of  claim 56  wherein the pseudo ITR has an imperfect palindomic DNA sequence. 
     
     
       58. The method of  claim 56  wherein the pseudo ITR has SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:8.

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